This invention relates to antibodies to argatroban and argatroban derivatives and their use in medicine. In particular, this invention relates to the field of detection of argatroban and argatroban derivatives and the inactivation, removal and/or sequestration of argatroban and argatroban derivatives using antibodies specific for argatroban and argatroban derivatives.
Maintenance of therapeutic agents at effective therapeutic levels is of critical importance in providing effective treatment for a host of conditions. In many treatment methods, effective therapeutic levels are only achieved by repetitive administrations of agents due to short half-lives of the particular agents in vivo. Often, large boluses of agent are administered, leading to initial concentrations above therapeutic levels, which may have adverse effects, followed by sub-optimal concentrations as the agent is degraded or cleared. U.S. Pat. No. 5,612,034 addresses this limitation by providing a method for prolonging the lifetime of an agent in the bloodstream by covalently linking derivatized agents to serum proteins and blood cells. The use of these derivatives presents new needs for analytical reagents to monitor the course of treatment. In addition, the use of these derivatives presents a need for means to inactivate, remove or sequester the derivatives and resulting conjugates during courses of treatment where toxicity or other adverse effects associated with the derivatives or resulting conjugates may be of concern.
As an example, the administration of the synthetic thrombin inhibitor argatroban: 1-[5-(Aminoiminomethyl)amino]-1-oxo-2-[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl]-amino]pentyl]-4-methyl-2-piperidinecarboxylic acid, is a preferred treatment of thrombosis. The efficacy of argatroban, as well as other thrombin inhibitors such as heparin, is limited by its short half-life in patients. In order to address this limitation, active derivatives of argatroban were synthesized whose half-life in the bloodstream is extended considerably compared to the parent molecule, as described in U.S. Pat. No. 5,840,733, incorporated herein by reference. The increase in persistence is due to the covalent linkage of reactive moieties of the argatroban derivatives to cells or stable serum proteins upon administration. These argatroban derivatives are favored because they enable administration of a more constant therapeutic level of anti-thrombin activity as compared to the available treatments, which require frequent administrations of large boluses of drug, followed by cycles of sub-optimal concentrations in the patient.
While these argatroban derivatives provide benefits in the treatment of thrombosis, their use presents new needs for analytical reagents to monitor the course of treatment. In particular, it is necessary to monitor the level of the derivatized argatroban molecule in vivo in order to adjust the dosage schedule to ensure maintenance of proper levels within the patient. Furthermore, the desirable increased stability of these argatroban derivatives in vivo might lead to new problems during treatment. Because argatroban-blood component conjugates are not rapidly cleared from the bloodstream, the possibility for toxicity is increased. Unlike heparin, whose toxic doses may be treated by protamine sulphate, there are no known antagonists to argatroban. Thus, there is also a need to identify a means to efficiently inactivate, remove or sequester argatroban conjugates from the blood stream in vivo in order to treat patients who suffer unacceptable amounts of bleeding or other side-effects associated with thrombolytic treatment.
The present invention is directed to isolated and purified antibodies that specifically bind argatroban and argatroban derivatives. The antibodies of this invention specifically bind argatroban derivatives including such derivatives as argatroban-C6-NHS, argatroban C-12-NHS, argatroban C-13 maleimide, argatroban C21-PE maleimide and argatroban C-18 maleimide.
The present invention is further directed to a kit for detecting the concentration of argatroban or argatroban derivatives in a solution such as a blood or serum sample comprising an antibody that specifically binds argatroban and argatroban derivatives. The antibody in the kit may be conjugated to an indicator reagent. The kit may further comprise reagents for performing immunoassays including ELISA, RIA, Western Blot, immunohistochemistry and flow cytometry reagents.
The present invention further relates to methods for determining the A concentration of an argatroban derivative and conjugates of argatroban in biological samples, in particular to methods that employ antibodies specific for the agent or its derivatives and conjugates. In addition, the invention relates to the use of these antibodies as a treatment for potential toxicity associated with stable argatroban conjugates. In one aspect of the invention, the invention relates to methods for determining the concentration of argatroban or its derivatives and conjugates in biological samples using antibodies specific to argatroban or its derivatives and conjugates, and to the use of such antibodies as a treatment for toxicity potentially associated with stable argatroban conjugates.
A third aspect of this invention relates to the use of monoclonal antibodies for the detection, separation and purification of diastereoisomeric mixtures of argatroban and argatroban derivatives.